[Show abstract][Hide abstract] ABSTRACT: Epstein-Barr virus (EBV) establishes a life-long latent infection in humans. In proliferating latently infected cells, EBV genomes persist as multiple episomes that undergo one DNA replication event per cell cycle and remain attached to the mitotic chromosomes. EBV nuclear antigen 1 (EBNA-1) binding to the episome and cellular genome is essential to ensure proper episome replication and segregation. However, the nature and regulation of EBNA-1 interaction with chromatin has not been clearly elucidated. This activity has been suggested to involve EBNA-1 binding to DNA, duplex RNA, and/or proteins. EBNA-1 binding protein 2 (EBP2), a nucleolar protein, has been proposed to act as a docking protein for EBNA-1 on mitotic chromosomes. However, there is no direct evidence thus far for EBP2 being associated with EBNA-1 during mitosis. By combining video microscopy and Förster resonance energy transfer (FRET) microscopy, we demonstrate here for the first time that EBNA-1 and EBP2 interact in the nucleoplasm, as well as in the nucleoli during interphase. However, in strong contrast to the current proposed model, we were unable to observe any interaction between EBNA-1 and EBP2 on mitotic chromosomes. We also performed a yeast double-hybrid screening, followed by a FRET analysis, that led us to identify HMGB2 (high-mobility group box 2), a well-known chromatin component, as a new partner for EBNA-1 on chromatin during interphase and mitosis. Although the depletion of HMGB2 partly altered EBNA-1 association with chromatin in HeLa cells during interphase and mitosis, it did not significantly impact the maintenance of EBV episomes in Raji cells.
Journal of Virology 02/2012; 86(9):5314-29. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Time-resolved microspectrofluorometry in live cells, based on time- and space-correlated single-photon counting, is a novel method to acquire spectrally resolved fluorescence decays, simultaneously in 256 wavelength channels. The system is calibrated with a full width at half maximum (FWHM) of 90 ps for the temporal resolution, a signal-to-noise ratio of 10(6), and a spectral resolution of 30 (Deltalambda/Lambda). As an example, complex fluorescence dynamics of ethidium and cyan fluorescent protein (CFP) in live cells are presented. Free and DNA intercalated forms of ethidium are simultaneously distinguishable by their relative lifetime (1.7 ns and 21.6 ns) and intensity spectra (shift of 7 nm). By analysing the complicated spectrally resolved fluorescence decay of CFP, we propose a fluorescence kinetics model for its excitation/desexcitation process. Such detailed studies under the microscope and in live cells are very promising for fluorescence signal quantification.
Journal of Microscopy 03/2004; 213(Pt 2):110-8. · 1.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Fluorescence anisotropy decay microscopy was used to determine, in individual living cells, the spatial monomer-dimer distribution of proteins, as exemplified by herpes simplex virus thymidine kinase (TK) fused to green fluorescent protein (GFP). Accordingly, the fluorescence anisotropy dynamics of two fusion proteins (TK27GFP and TK366GFP) was recorded in the confocal mode by ultra-sensitive time-correlated single-photon counting. This provided a measurement of the rotational time of these proteins, which, by comparing with GFP, allowed the determination of their oligomeric state in both the cytoplasm and the nucleus. It also revealed energy homo-transfer within aggregates that TK366GFP progressively formed. Using a symmetric dimer model, structural parameters were estimated; the mutual orientation of the transition dipoles of the two GFP chromophores, calculated from the residual anisotropy, was 44.6 +/- 1.6 degrees, and the upper intermolecular limit between the two fluorescent tags, calculated from the energy transfer rate, was 70 A. Acquisition of the fluorescence steady-state intensity, lifetime, and anisotropy decay in the same cells, at different times after transfection, indicated that TK366GFP was initially in a monomeric state and then formed dimers that grew into aggregates. Picosecond time-resolved fluorescence anisotropy microscopy opens a promising avenue for obtaining structural information on proteins in individual living cells, even when expression levels are very low.
[Show abstract][Hide abstract] ABSTRACT: The effects of various degrees of perturbation of the mitochondrial membrane potential (mt delta psi) on apoptosis was investigated by intensified fluorescence digital-imaging microscopy on neuron-like cells, ND7. Mt delta psi was either decreased by 40% by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP 100 nM, 15 min) or completely collapsed (FCCP 10 microM, 60 min). A moderate decrease of mt delta psi induced a reduction of mitochondrial NADH, followed by exposure of phosphatidyl serine and then by chromatin condensation, 36% of nuclei being condensed 60 min after FCCP treatment. During these stages, mitochondrion morphology was fully preserved. In contrast, no chromatin condensation was observed after a rapid and total dissipation of mt delta psi. These results suggest that a partial decrease of mt delta psi would allow mitochondrial functions required to trigger apoptosis to be sustained.
[Show abstract][Hide abstract] ABSTRACT: Physical parameters, describing the state of chromatinized DNA in living mammalian cells, were revealed by in situ fluorescence dynamic properties of ethidium in its free and intercalated states. The lifetimes and anisotropy decays of this cationic chromophore were measured within the nuclear domain, by using the ultra-sensitive time-correlated single-photon counting technique, confocal microscopy, and ultra-low probe concentrations. We found that, in living cells: 1) free ethidium molecules equilibrate between extracellular milieu and nucleus, demonstrating that the cation is naturally transported into the nucleus; 2) the intercalation of ethidium into chromatinized DNA is strongly inhibited, with relaxation of the inhibition after mild (digitonin) cell treatment; 3) intercalation sites are likely to be located in chromatin DNA; and 4) the fluorescence anisotropy relaxation of intercalated molecules is very slow. The combination of fluorescence kinetic and fluorescence anisotropy dynamics indicates that the torsional dynamics of nuclear DNA is highly restrained in living cells.
[Show abstract][Hide abstract] ABSTRACT: The effect of controlled and reversible perturbation of the electrochemical gradient on the structural changes of mitochondrial DNA has been studied in living cells by fluorescence microscopy. Electrochemical gradient perturbations were induced by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone and quantified by measuring the mitochondrial membrane potential using tetramethyl rhodamine methyl ester. Under our experimental conditions, we have shown that ethidium fluorescence was mainly due to ethidium molecules intercalated in mtDNA. Ethidium fluorescence variations have been used to probe DNA structural changes. This showed that: i) electrochemical gradient perturbations induced mtDNA structural change; ii) this change was readily reversible following a total but short collapse of the electrochemical gradient; iii) in contrast, a short and weak perturbation of the electrochemical gradient stabilized the mtDNA structural change; and iv) the degree of weak depolarization varied from cell to cell, showing the necessity of studying the effect of energetic perturbations at the level of an individual cell.
Biology of the Cell 12/1999; 91(8):597-604. · 3.49 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Epstein-Barr virus (EBV) genome can persist in dividing human B cells as multicopy circular episomes. Viral episomes replicate in synchrony with host cell DNA and are maintained at a relatively constant copy number for a long time. Only two viral elements, the replication origin OriP and the EBNA-1 protein, are required for the persistence of viral genomes during latency. EBNA-1 activates OriP during the S phase and may also contribute to the partition and/or retention of viral genomes during mitosis. Indeed, EBNA-1 has been shown to interact with mitotic chromatin. Moreover, viral genomes are noncovalently associated with metaphase chromosomes. This suggests that EBNA-1 may facilitate the anchorage of viral genomes on cellular chromosomes, thus ensuring proper partition and retention. In the present paper, we have investigated the chromosome-binding activity of EBV EBNA-1, herpesvirus papio (HVP) EBNA-1, and various derivatives of EBV EBNA-1, fused to a variant of the green fluorescent protein. The results show that binding to metaphase chromosomes is a common property of EBV and HVP EBNA-1. Further studies indicated that at least three independent domains (CBS-1, -2, and -3) mediate EBNA-1 binding to metaphase chromosomes. In agreement with the anchorage model, two of these domains mapped to a region that has been previously demonstrated to be required for the long-term persistence of OriP-containing plasmids.
Journal of Virology 06/1999; 73(5):4385-92. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Digital-imaging microscopy was used in conditions that allowed the native state to be preserved and hence fluorescence variations of specific probes to be followed in the real time of living mammalian cells. Ethidium bromide was shown to enter into living cells and to intercalate stably into mitochondrial DNA (mtDNA), giving rise to high fluorescence. When the membrane potential or the pH gradient across the inner membrane was abolished by specific inhibitors or ionophores, the ethidium fluorescence disappeared from all mtDNA molecules within 2 min. After removal of the inhibitors or ionophores, ethidium fluorescence rapidly reappeared in mitochondria, together with the membrane potential. The fluorescence extinction did not result from an equilibrium shift caused by leakage of free ethidium out of mitochondria when the membrane potential was abolished but was most likely due to a dynamical mtDNA change that exposed intercalated ethidium to quencher, either by weakening the ethidium binding constant or by giving access of a proton acceptor (such as water) to the interior of mtDNA. Double labeling with ethidium and with a minor groove probe (4',6-diamino-2-phenylindole) indicated that mtDNA maintains a double-stranded structure. The two double-stranded DNA states, revealed by the fluorescence of mitochondrial ethidium, enhanced or quenched in the presence of ethidium, seem to coexist in mitochondria of unperturbed fibroblast cells, suggesting a spontaneous dynamical change of mtDNA molecules. Therefore, the ethidium fluorescence variation allows changes of DNA to be followed, a property that has to be taken into consideration when using this intercalator for in vivo as well as in vitro imaging studies.
[Show abstract][Hide abstract] ABSTRACT: Apoptosis, related to a naturally-occurring or programmed cellular death process, can be physiologically or exogenously induced. In vertebrate cells undergoing apoptosis, initiated by any of these ways, one of the numerous biochemical changes is an endogenous endonuclease activation that cleaves the chromatin DNA into oligonucleosome-sized 'ladder' fragments. In the present study we show that in parallel to chromatin DNA cleavage, ribosomal RNA is lost in gamma-ray-mediated apoptotic human lymphocytes. We demonstrate that 28S rRNA gene transcription is induced early (15 min) after irradiation, followed by a selective disappearance in apoptotic cells only. The fact that newly synthesized rRNA turns over at the same rate in irradiated and untreated cell fractions, highly suggests that the observed loss of 28S rRNA in the apoptotic cell fraction at the ribosome level is due to degradation occurring at a late stage of the apoptotic death process. These results suggest that, in addition to first-stage apoptosis-associated rDNA gene activation, cellular self-destruction at late stages is associated with processes occurring simultaneously at the ribosome level involving an endogenous RNase-like activity, and at the chromatin level involving DNA-nuclease activity.
International Journal of Radiation Biology 08/1993; 64(1):39-46. · 1.84 Impact Factor